Liposomal formulations of therapeutically active drugs have significant advantages over drugs injected in free form. Weinstein, Liposomes: From Biophysics to Therapeutics, (Ostro, M. J., ed.), Marcel Dekker, Inc., N.Y., pp. 277-338, (1987). For example, liposomal formulations of the anti-cancer alkaloid vincristine have greater efficacy against L1210 leukemia cells than does free vincristine and have reduced collateral toxicity. Mayer et al., Cancer Chemother. Pharmacol. 33:17-24 (1993) and Mayer et al., Cancer Res. 50:575-579 (1990). The development of liposomal formulations of therapeutic agents with clinical and/or pharmaceutical potential depends on the liposome/drug combination possessing both biological efficacy and long-term chemical stability. In general, the efficacy of a liposomal agent can be improved by increasing both the liposome circulation lifetime and the ability of the liposome to retain the encapsulated drug. Mayer, ibid, and Boman et al., Cancer Res. 54:2830-2833 (1994). Therefore, much effort has focused on the development of liposomal formulations of therapeutic compounds having both extended circulation times and enhanced drug retention.
A wide variety of therapeutic agents can be loaded into liposomes with encapsulation efficiencies approaching 100% by using a transmembrane pH gradient. Mayer et al., Biochim. Biophys. Acta 1025:143-151 (1990) and Madden et al., Chem. Phys. Lipids 53:37-46 (1990). The chemical stability of these formulations, i.e., the effective retention of the loaded drugs within the liposomes during circulation in vivo, frequently requires that the intraliposomal pH be in the range between pH 2.0 to 4.0. Within this pH range however, acid hydrolysis of the acyl component of liposomes can destabilize the liposomal membranes and result in premature leakage of the drug.
For example, vincristine can be loaded efficiently into liposomes by a pH gradient-dependent encapsulation procedure which employs an intraliposomal pH of 4.0. Mayer et al., Biochim. Biophys. Acta 1025:143-151 (1990) and Mayer et al., Cancer Res. 50:575-579 (1990). The work with liposomal vincristine has been based on vesicles containing phosphatidylcholine (PC), usually egg PC or distearoyl-PC, and cholesterol. Mayer et al., 1993, supra. Increased anti-tumor efficacy of liposomal vincristine occurs when the in vivo retention of vincristine in the liposomes is increased using a 100-fold larger transmembrane pH gradient (i.e. intraliposomal pH=2.0). Boman et al., supra. However, at this pH the acid-hydrolysis of the PC component of the liposomes occurs at a significant rate and severely limits the chemical stability of the liposomes. In particular, the fatty acid carboxyl esters at positions sn-1 and sn-2 are especially susceptible to acid-hydrolysis to produce lyso-PC and free fatty acids. Grit et al., Chem. Phys. Lipids 64:3-18 (1993). Liposomes containing significant proportions of lyso-PC are more permeable to solutes, and therefore would be unsuitable as drug delivery vehicles.
It has been reported that sphingomyelin imparts an increase in the circulation lifetime of liposomes. Allen et al., Biochim. Biophys. Acta 981:27-35 (1989) and Allen et al., FEBS Lett. 223:42-46 (1987). However, these studies employed an entrapped aqueous solute (.sup.125 I-tyraminylinulin) as a marker for liposome distribution, and the apparent increase in liposome longevity in the presence of sphingomyelin may have resulted at least in part from increased solute retention by sphingomyelin. There have also been several reports that sphingomyelin-containing liposomes are more toxic than PC-containing liposomes. Weereratne et al., Brit. J. Exp. Pathol. 64:670-676 (1983), Allen et al., J. Pharmacol. Exp. Therap. 229:267-275 (1984), and Allen et al., Res. Commun. Chem. Pathol. Pharmacol. 50:281-290 (1985). Although more conclusive studies are not available, the perception is that sphingomyelin-containing liposomes are associated with an increased risk of toxicity.
Liposomal formulations of therapeutic compounds having increased biological and chemical stability are needed in the art. As the efficacy of liposomal agents may be improved by increasing the liposome circulation time and the ability of the liposome to retain the encapsulated drug, the development of liposomal formulations having these properties would be valuable additions to clinical treatment regimens. Quite surprisingly, the present invention fulfills these and other related needs.